Studies On Mitochondrial Genome Differences Of Monozygotic Twins Based On The Next-generation Sequencing

Objective: In the actual forensic cases,if the on-site biological samples are left by monozygotic twins(MZT),the current forensic DNA techniques cannot yet effectively distinguish which individual of MZT is the real one who left the samples due to the same genetic sequences of them.This leads to such case difficult or even impossible to be resolved and brings uncertain factors to identify the real criminal.We have conducted a series of studies on this issue and focused on the epigenetic differences between MZT.We found that there are differences of DNA methylation between MZT through whole genome DNA methylation analysis,and we selected 9 sequences with 39 CpG sites,after validation using large samples of MZT by pyrosequencing,to build a detection system to distinguish MZT,the power of which reached to 94.2%.However,DNA methylation is not as stable as DNA sequences,which might be changed with age or environment.It also requires higher quality and quantity of DNA to measure DNA methylation.So these factors might limit the application of DNA methylation in the actual forensic cases.It was reported that there are little DNA sequence differences in the nuclear genome between MZT,but finding the sequence difference is like finding a needle in a haystack.And the cost of whole genome sequencing is relatively high and data analysis is an extremely tough work.So it is obviously not applicable for forensic cases.Mitochondrial DNA(mtDNA),only 16,569 bp,with higher mutation rate than nuclear genome due to exposure to oxidation reactions and without repair system,may be a better candidate genetic marker for discriminating MZT.Theoretically,after isolated from embryos,MZT have a greater probability of accumulating different mtDNA mutations in the process of growth and development.So there are more chances to find mtGenome differences between MZT.Moreover,the cost of mtDNA genome sequencing and data analysis is greatly reduced.The cyclic structure of mtDNA makes it highly resistant to degradation,and the large copy numbers of mtDNA also enhance the detection sensitivity of it.In recent years,the development of next generation sequencing(NGS)technology provides a powerful tool for mtGenome sequencing.Therefore,in this study,we intends to use NGS technology to detect the entire mtGenome sequence of 21 MZT pairs,to observe whether there are differences in mtDNA sequence between MZT and evaluate the potential application value in forensic science,so as to provide scientific basis for solving the MZT distinguishing problems.Methods:1 Sample collection and DNA quantification: From the twin database established by our research group,we selected 21 pairs of MZT’s whole genome DNA samples.We extracted DNA from the hair follicles and hair shafts of volunteers(FSY,TL)using the QIAamp DNA Investigator Kit.All DNA samples were quantified using the Qubit? ds DNA HS Assay Kit and sample 149 b was quantified absolutely using the Quantifiler? Human DNA Quantification Kit and the 7500 Real-time PCR System.2 Precision ID mt DNA Panel laboratory evaluation: The library of 100 a,100b and 9947 A standard were constructed and sequenced in triplicate in repeatability studies.After absolute quantification,the sample 149 b was employed as a series of input DNA(100 pg,50 pg and 25 pg)in duplicate in sensitivity studies.The library of hair follicles and hair shafts were constructed and sequenced in specificity studies.We sequenced the mtGenome of the samples 137 a and 137 b using PGM and MiSeq platform for concordance studies.3 Library Construction: We prepared libraries using 0.1 ng of DNA with the Precision ID mtDNA Panel and Precision ID Library Kit according to the manufacturer’s protocol.4 Template Preparation: Sequencing template was prepared using the Ion PGM? Hi-Q? OT2 Kit on Ion OneTouch? 2 System according to the manufacturer’s protocol.5 Ion PGM? sequencing: Samples were sequenced on Ion PGM? System using Ion PGM? Hi-Q? Sequencing Kit according to the manufacturer’s protocol.6 Results Analysis: We analyzed mtGenome sequencing results using the Ion Torrent Software Suite with coverageAnalysis and Torrent Variant Caller(TVC)referred to rCRS(GenBank ID: NC-012920.1).The repeatability,sensitivity,specificity and concordance studies were statistically analyzed,and the performance in the laboratory was evaluated comprehensively.We looked for the differences of MZT by comparing the point heteroplasmy and length heteroplasmy.MtDNA haplotypes were determined on EMPOP.7 We used Sanger sequencing technology to verify the heteroplasmy.Results:1 Precision ID mtDNA Panel laboratory evaluation1.1 Coverage depthIn this study,we detected the mtGenome of 49 samples(from 46 individuals and 9947 A standard).The average sequencing depth was 2433×(± 205×)(mean ± SE).1.2 Repeatability studyTwo samples(100a and 100b)were used to construct three libraries and sequenced on the same chip.Two libraries were constructed using 9947 A standard DNA and sequenced on two chips.There was no significant difference between the replicates.1.3 Sensitivity studyThe mt DNA was still completely detected with a low input DNA amount of 25 pg,and the coverage was good.1.4 Specificity studyIn this study,by comparing the TVC results of hair follicles and hair shafts from the same individual,we found that the variants and frequency of the two samples were consistent,indicating the mt DNA sequence we detected was specific without the contamination of nuclear mitochondrial pseudogenes(NUMTs).1.5 Concordance studyBy comparing the sequencing results of PGM platform and Mi Seq platform,only #5951 site was different,and the other sites were the same.Therefore,this method shows good consistency in our laboratory.2 mtDNA sequence and heteroplasmy analysis2.1 mtDNA sequence analysisCompared with revised Cambridge Reference Sequence(rCRS),a total of 1917 variants were observed at 311 nucleotide positions from 46 individuals and 9947 A standard,including 545 variants(28.43%)in the control region(CR)and 1372 variants(71.57%)in the coding region.Distribution of variants in the CR is denser.About the variants,87.38% were substitutions and 12.62% were indels.Among them,variants 263 G,750G,4769 G,8860G and 15326 G were observed in all 47 samples.Variants 73 G,2706T,7028 T and 11719 A were observed in 46 samples,excepted 9947 A.9947A roots in Caucasian,so the sequence differences of these 4 positions reflect the differences between Chinese and Caucasian populations.2.2 mtDNA heteroplasmy analysisAccording to the system setting,the heteroplasmy detection threshold of TVC result was 10%,a total of 14 PHPs and 42 LHPs were detected in the 21 pairs of MZT.Among them,PHPs were found in 8 samples,and LHPs were found in 24 samples,and no heteroplasmy was found in 24 samples.Most of these MZT share the same heteroplasmy.PHP 8495A/C was present in 2 pairs of MZT,LHP 3492.1A was present in 7 individuals of 4 pairs of MZT,LHP 13797.1A was present in 2 pairs of MZT,and both 10873 C and LHP 10873 DEL were present in 4 pairs of MZTs.3 mtDNA difference analysis of MZTNo difference of sequence variation was observed in the 21 pairs of MZT,but only differences on heteroplasmy level were observed.Although most of MZT share the same heteroplasmy,but the minor allele frequency of heteoplasmy was found to be different for some of them.We set 10% and 15% as mtDNA heteroplasmy difference analysis thresholds.Among the 21 pairs of MZT detected,there are 9 pairs with heteroplasmy frequency difference ≥10%,including 2 pairs with PHP frequency difference ≥10% and 8 pairs with LHP frequency difference ≥10%.That indicated with the standard of mt DNA heteroplasmy frequency difference ≥10%,the two kinds of heteroplasmy can distinguish 9 pairs of MZT,accounting for 42.86%.There are 2 pairs with heteroplasmy frequency difference ≥15%,including 1 pair with PHP frequency difference ≥15% and 2 pairs with LHP frequency difference ≥15%,which means with the standard of mtDNA heteroplasmy frequency difference ≥15%,the two kinds of heteroplasmy can distinguish 2 pairs of MZT,accounting for 9.52%.4 HaplogroupsThe haplogroups of 47 samples were confirmed and determined on EMPOP.23 different haplogroups were assigned to M,B,D,F,C and H respectively 27.66%,25.53%,25.53% 14.89%,4.26% and 2.13%.MZT share the same haplotype.5 The correlation between heteroplasmy and ageWe analyzed the correlation between the number of heteroplasmy of 46 individuals and age,and the results showed that there is no correlation between the number of heteroplasmy and age.Conclusions:In this study,we detected whole mitochondrial genome of 21 pairs of MZT using Precision ID mtDNA Panel on the Ion Torrent PGM? platform.It was found that there was no difference in the level of sequence variability between MZT,but there were differences in the heteroplasmy level.There were 9 pairs of MZT with heteroplasmy frequency difference ≥10%,including 2 pairs of MZT with heteroplasmy frequency differences ≥15%.Therefore comparing mtGenome of MZT using the NGS technology provides a new solution for distinguishing MZT,which exhibits specificity without contamination of NUMTs,repeatability,stability and high sensitivity.In the following study,systematic forensic application evaluation will be conducted to further verify the practical application value of this technique.